A hepatitis C virus (hereinafter may be referred to as “HCV”) is the main virus causing post-transfusion non-A, non-B hepatitis, and has a positive-strand RNA genome of about 9,500 bases. According to statistics from Ministry of Health, Labour and Welfare, it is estimated that there are 1.5 million or more HCV carriers in Japan in 2000. Patients infected with the HCV have a strong tendency to proceed to chronic hepatitis, hepatic cirrhosis, or hepatic cancer via transient acute hepatitis. It is believed that about 80% of primary hepatic cancer in Japan is HCV-related hepatic cancer. Genotypes of the HCV are classified according to homology of base sequences. There are about 30 genotypes all over the world. In Japan, five genotypes, i.e., type 1a, type 1b, type 2a, type 2b, and type 3a, have been found to infect Japanese or have been detected in Japan. In Japan, types 1b, 2a, and 2b account for most of the five genotypes, while there are few viruses in types 1a or 3a.
Interferons (hereinafter may be referred to as “IFN”) have been used for treating chronic hepatitis due to the HCV. Direct-acting antiviral agents have also been used.
An example of the direct-acting antiviral agents includes daclatasvir. The daclatasvir is an inhibitor of an HCV NS5A (non-structural 5A) protein. It has been found that a mutation in an amino acid at position 93 (wild type: tyrosine) of the HCV NS5A protein causes resistance to the daclatasvir (see, e.g., NPL 1). Therefore, there is a need to verify whether the amino acid at position 93 of the HCV NS5A protein is mutated prior to administration of the daclatasvir to patients at a low cost, easily, conveniently, and rapidly.
It has been known that a plurality of HCV variants are mixedly present in a human body. Therefore, when verifying whether the amino acid at position 93 of the HCV NS5A protein in a sample is mutated, there is also a need to quantify HCVs with and without the mutation and determine a mutation rate thereof in addition to the presence or absence of the mutation.
Examples of a method for verifying the presence or absence of the mutation include a direct sequencing method and sequencing with a next-generation sequencer.
Although the direct sequencing method can be inexpensively performed, it is not quantitative. Therefore, there is a problem that it is impossible to quantify a wild-type and a mutated form or to determine the mutation rate thereof by the direct sequencing method. Meanwhile, although the sequencing with a next-generation sequencer is quantitative, a cost per run is very huge. Therefore, there is a problem that it is virtually impossible to use for HCV which is rarely examined in clinical practice and of which genome has a relatively small size.
The HCV is prone to mutation. It has been known that a single genotype includes many variants with a difference of 1 to several hundred bases. Therefore, there is a need for a method for verifying whether the amino acid at position 93 of the HCV NS5A protein in a sample is mutated while suppressing an influence of mutations or variations in the HCV, the method can be applied to various HCVs in the sample.
Therefore, at present, keen demand has arisen for quickly developing a method for quantitatively measuring the presence or absence of a mutation in an amino acid at position 93 of various HCV NS5A proteins in a sample at a low cost, easily, conveniently, and rapidly while suppressing an influence of mutations or variations in HCV.